How is depth of anaesthesia assessed in experimental pigs? A scoping review

Background Despite the large number of pigs involved in translational studies, no gold standard depth of anaesthesia indicators are available. We undertook a scoping review to investigate and summarize the evidence that sustains or contradicts the use of depth of anaesthesia indicators in this species. Methods Medline, Embase and CAB abstract were searched up to September 22nd 2022. No limits were set for time, language and study type. Only original articles of in vivo studies using pigs or minipigs undergoing general anaesthesia were included. The depth of anaesthesia indicators reported in the selected papers were divided in two categories: A, indicators purposely investigated as method to assess depth of anaesthesia; B, indicators reported but not investigated as method to assess depth of anaesthesia. Results Out of 13792 papers found, 105 were included after the screening process. Category A: 17 depth of anaesthesia indicators were found in 19 papers. Studies were conducted using inhalant anaesthetics as the main anaesthetic agent in the majority of the cases (13/19 = 68.4%), while 3/19 (15.8%) used propofol. The most investigated depth of anaesthesia indicators were bispectral index (8/19 = 42.1%) and spectral edge frequency 95% (5/19 = 26.3%). Contrasting results about the specific usefulness of each depth of anaesthesia indicators were reported. Category B: 23 depth of anaesthesia indicators were found in 92 papers. The most reported depth of anaesthesia indicators were: motor response following a stimulus (37/92 = 40.2%), depth of anaesthesia scores (21/92 = 23.3%), bispectral index (16/92 = 17.8%) and spectral edge frequency 95% (9/92 = 9.8%). Conclusion Results highlight the lack of scientifically valid and reliable indicators to ensure adequate depth of anaesthesia in pigs.


Introduction
The porcine model is extensively used in translational medicine due to the anatomical and physiological similarities between humans and pigs [1][2][3][4]. The last European Union's Report on animal research estimated that between 2015 and 2017 around 80000 pigs were used yearly in biomedical research [5]. Despite the large number of animals involved in translational studies, no "gold standard" methodology has been defined to assess anaesthetic depth. The impact of this problem is even more pronounced when neuromuscular blocking agents (NMBAs) are administered [6,7]. This is not only a serious ethical concern, but it raises questions about the reliability of the results obtained from translational studies, especially those assessing perioperative cardiovascular, respiratory and hormonal parameters.
We undertook a scoping review to investigate and synthetize the evidence that sustains or contradicts the use of the depth of anaesthesia (DoA) indicators reported in the literature and adopted in pigs and minipigs undergoing general anaesthesia. In order to reach this goal, the following research question was defined: "Is there scientific evidence of the usefulness of the methodologies commonly employed to assess DoA in pigs?".
A scoping review was judged appropriate to provide a comprehensive and thorough overview of the available literature in the broad subject of interest.
The aims of the present work were: • To identify the indicators that have been investigated to assess DoA in pigs; • To investigate and summarize the evidence that sustains or contradicts the use of DoA indicators reported in the literature and adopted in pigs and minipigs undergoing general anaesthesia; • To identify knowledge gaps.

Materials and methods
The present scoping review was conducted following the PRISMA Extension for Scoping Reviews (PRISMA-ScR) guidelines [8]. A protocol, adapted from the PRISMA-P guidelines [9] was developed a priori (see supplementary data). The search strategies were developed in collaboration with the systematic review services coordinator of the University of Bern.

Stage 1: Identification of DoA indicators
Phase 1: an electronic literature search was performed on January 20 th 2020 by a single investigator (AM) using three databases (PubMed, Embase, CAB abstract) via Ovid, to develop a preliminary list of DoA indicators reported in pigs (material available from the author upon request). No time, language and study type limitations were set. The identified citations were imported into the Mendeley Reference Management Software and duplicates were removed. Title, abstract and keywords were screened to identify DoA indicators. In case it was not clear in which species a certain DoA indicator was used, it was included in the list. Moreover, additional searches were performed via Google Scholar. Phase 2: an initial list was developed including all the DoA indicators found during phase 1. This list was provided to anaesthetists of the Anaesthesiology and Pain Therapy Section, Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, who were asked to check it and add potential missing items.
Phase 3: the developed list was sent world-wide through the veterinary anaesthesia list server (ACVA-List) to veterinary anaesthesia specialists, with the same request. The final list including all DoA indicators collected from phases 1, 2 and 3 was established and used for the next step (Stage 2).

Stage 2: Search strategy
Based on the list settled in Stage 1, a search strategy was developed (see supplementary data). It aimed at identifying papers including pigs undergoing general anaesthesia and where at least one of the previously listed DoA indicators was reported.
The electronic literature search was performed using the same aforementioned databases via Ovid: PubMed, Embase, CAB abstract. The first search was performed on March 19 th 2020, and then updated on September 22 nd 2022. No time, language and study type limitations were set. The identified citations were imported into Mendeley Reference Management Software and duplicates were removed.

Stage 3: Paper selection
Two independent investigators (AM, EGM) screened title and abstract of papers identified through the first search (until March 19 th 2020) and selected those that met all the following four inclusion criteria: 1. reported original research; 2. performed using pigs or minipigs; 3. performed in vivo; 4. involved animals undergoing general anaesthesia.
The investigators periodically met to compare screening results. Differences were discussed and resolved by consensus. In case one of the aforementioned points was not evident when reading title and abstract, the paper was included for further screening.
As the selection identified a large amount of papers, a further inclusion criterion (i.e. that an indicator of DoA must be mentioned in the title or abstract) was added and applied by a single investigator (AM).
For the search update (from March 19 th 2020 to September 22 nd 2022), papers were selected by a single investigator (AM) applying the five inclusion criteria.

Stage 4: Full text screening
The full-text screening was performed by a single investigator (AM). In case of doubt, a second investigator (CS or OL) was asked to screen the text independently, and the final result was obtained upon consensus. During this phase, papers were excluded if: • the authors did not report any DoA indicator; • the DoA indicators were not reported for at least two well distinct time points and/or drug combinations and/or drug concentrations and/or physiological conditions and/or surgical interventions; • unconsciousness was not pharmacologically induced (e.g., electricity, carbon dioxide); • only a minimum alveolar concentration (MAC) extrapolated from other studies was used to guide anaesthetic administration; • the paper was found to be a duplicate, or at least one of the five abstract inclusion criteria was not fulfilled; • only the abstract was available.
From the included papers, DoA indicators were identified and categorized as follows: • category A: indicators purposely investigated as method to assess depth of anaesthesia; • category B: indicators reported but not investigated as method to assess depth of anaesthesia Electrical nociceptive stimuli were also applied in isoflurane anaesthetized pigs to evoke a nociceptive withdrawal reflex (NWR). Reflex amplitude decreased with increasing isoflurane concentrations, and reflexes evoked by single stimuli were suppressed at lower concentrations than those evoked by repeated stimuli, showing that temporal summation still occurs around

PLOS ONE
How is depth of anaesthesia assessed in experimental pigs? A scoping review MAC levels [23]. In a recent study, the NWR was shown to be influenced in a nonuniform manner by different drugs administered at increasing doses, and to give dissimilar results when compared to response to claw clamp [26]. Category B. All the studies focused on pigs and were published between 1980 and 2022. The most reported DoA indicator was the appearance of a motor response following a stimulus (37/92 = 40.2%), that was nociceptive in 34/92 cases (37%). It was followed by DoA scores (21/92 = 22.8%), BIS (16/92 = 17.4%) and SEF 95% (9/81 = 9.8%).

Discussion
The present scoping review highlights the lack of scientifically valid methods to ensure an adequate DoA in pigs undergoing general anaesthesia. Only few investigations have been performed for this purpose. Moreover, heterogeneous and contrasting results about the usefulness of the DoA indicators used has been found, making difficult to draw final conclusions. Furthermore, inhalant anaesthetics have been mainly administered, and almost no information is present regarding DoA assessment during total intravenous anaesthesia. Accidental awareness is one of the main consequences of an inadequate DoA. In humans, its incidence has been reduced from 1-2% in the 1980s to around 0.1% nowadays [7, 115,116]. It has been previously highlighted that accidental awareness is likely to be prevalent in pigs undergoing experimental studies, mainly caused by inadequate monitoring and poorly trained staff [6]. This is in line with our results showing that, currently, no valid monitoring strategies are available, leading to an increased risk of accidental awareness. Such a lack, on one side impedes the development of DoA assessment guidelines, on the other side represents a great ethical issue of which the scientific community should be aware.
Consciousness vanishes when anaesthetics produce loss of cortical connectivity and block the brain's ability to integrate information. Following this principle, in the last two decades,   Heart rate At a stable isoflurane concentration, piglets having a positive response to toe pinch showed a significant increase in heart rate but not in blood pressure.
However, the receiver operating characteristic (ROC) analysis revealed that heart rate was not a sensitive predictor of toe pinch motion response. Both before and after toe pinch, BIS bmean was a strong predictor of a positive motion response. , only single reports could be found; both indexes seemed to mirror the clinical judgement in animals either receiving a single propofol bolus or undergoing an isoflurane-based balanced anaesthesia, respectively. Further studies are needed to confirm these preliminary results. On the other side, the BIS has been assessed in eight studies (seven of which using inhalants) and contrasting results were reported. Greene and colleagues [11] and Haga and colleagues [12] found that, at increasing doses of isoflurane, the BIS remained stable, not providing any help

PLOS ONE
How is depth of anaesthesia assessed in experimental pigs? A scoping review in differentiating anaesthetic levels. These results are in contrast with those of Martín-Cancho and colleagues that showed how BIS could reflect increasing concentrations of isoflurane, sevoflurane and desflurane [14,16]. Schmidt and colleagues [17] reported similar BIS values while halothane, xenon or pentobarbital were administered, but various levels of DoA were not compared. Jaber and colleagues [13] reported that BIS can possibly be used to predict movements in response to noxious stimulation, in contrast with the results found by Baars and colleagues [10]. If the BIS has been developed to linearly follow an increasing DoA or rather to provide a range of values corresponding to an adequate DoA is not known. Indeed, the algorithm through which the EEG signal is processed is proprietary, and conclusions are difficult to be drawn.
It is also important to highlight that BIS, IoC and PSI have been developed based on data collected from humans, and a mere translation of their application in pigs could lead to misleading results. Species-specific anatomical peculiarities should be considered [19]. Furthermore, age, drugs used, clinical status, among others, have also been demonstrated to influence the EEG signal, in both humans and pigs [21,22,117,118] but are often not taken in consideration by the EEG-based DoA monitors developed so far. Further studies should be conducted to assess the validity of the BIS and other indexes in assessing DoA in pigs, using various anaesthetic regimens.
Other features extrapolated from the EEG have been applied to assess DoA in this species. Among them, SEF 95% and MED have been mostly reported. They represent the frequency below which 95% and 50% of the EEG total power is located, respectively. Both parameters were found not capable to correctly predict DoA in pigs. Moreover, they were largely influenced by the type of anaesthetic used, as well as by the presence of EEG suppression, similarly to what reported in humans [119][120][121]. Also in this case, no studies using total intravenous anaesthesia have been performed. Further investigations should be carried out before drawing final conclusions.
The use of EEG suppression to reach an adequate DoA has also been reported in pigs, but never thoroughly investigated. The suppression ratio represents the percentage of EEG activity that does not exceed a user-specified amplitude threshold (usually ± 5 μV) over a certain time span (usually 60-63 seconds) [122,123]. No specific end-points have been established in pigs, but thresholds values of 20% or above has been targeted to reach deep anaesthesia. Recent studies in humans [123,124] found the intraoperative presence of EEG suppression to be an independent risk factor for postoperative delirium. Despite no strong causative effect has been proved, and no information is available on the influence that EEG suppression might have on recovery outcomes in veterinary species, care should be taken in titrating drug administration only based on EEG suppression levels. Moreover, as previously mentioned, caution should be used in quantifying EEG suppression via DoA monitors whose algorithms have been created for humans.
Despite the mechanism of action of general anaesthetics has not been fully understood, their action is observed both at cortical and subcortical levels [125][126][127]. While the consciousness is mainly based on cortical neuronal networks, nociception and related motor responses are rather processed at subcortical levels [128][129][130][131][132].
Assessing whether a nociceptive stimulus is able to evoke a reaction or not was found to be the method mostly used to evaluate DoA in pigs. On responsiveness to stimulation is based the concept of MAC, namely the minimum alveolar concentration of anaesthetic at 1 atmosphere, which produces immobility in 50% of subjects exposed to a noxious stimulus [133]. It has often been used as a guide for inhalant drugs administration in many animal species. However, if consciousness is a state in which an individual is able to process information from his surroundings [134], MAC seems to be mainly related to spinal cord excitability rather than to consciousness [128][129][130][131][132]. This is in line with studies in humans, finding patient's responsiveness and consciousness to be not always related to each other [135,136]. Thus, the use of MAC as sole method to assess DoA should be avoided or at least its limitations clearly recognised.
The electrical NWR has also been investigated as DoA indicator. Even if this methodology has been principally applied to assess antinociception, studies support its possible use as adjunct in the DoA assessment [10,23]. Further investigations should be carried out to establish the most appropriate recording paradigms in this species, and to evaluate the effect that different drugs have on it.
Depth of anaesthesia scores have been widely used in the literature in pigs. However, none has been validated, and the scoring has been often based on the investigator subjective judgment. Despite being easy to apply, care should be taken when assessing DoA using scores which have not undergone a validation process.
Great effort has been put in the last years for the development, diffusion and promotion of the 3Rs principle [137][138][139]. One of its three cardinal points is the "refinement", namely the minimisation of pain, suffering, distress or lasting harm that may be experienced by research animals. In spite of that, only two studies have been performed to investigate DoA indicators in pigs in the last seven years.
All the pigs included in the selected papers were younger than 6 months of age. Even if juvenile animals are commonly employed in experimental settings due to the ease of handling, no information is currently available on any DoA indicator in adult and geriatric pigs. Care should be taken while translating the information currently available on DoA assessment to animals of different ages.
No information has been found in minipigs, despite their increasing use in different branches of translational medicine [140][141][142][143]. Further specific studies should be performed characterizing the effects induced by anaesthetic drugs in these animals, also considering that most of them are used as adult.
Considering the nature of our research question, a scoping review was deemed the most appropriate design. Due to the heterogeneity in the included studies, no risk of bias assessment was performed. Our results can be the basis for future focused systematic reviews, aiming at answering narrower and more specific questions and analysing the quality of individual studies.
Our scoping review has some limitations. First, due to limited personnel availability, not all the screening steps were performed by two persons. Despite the guidelines do not establish a minimum number of investigators [8], the involvement of more than one independent reviewer during the whole process would have guaranteed a higher level of reproducibility. Second, despite following a rigorous methodology, it is possible that relevant studies were missed during abstract selection if neither the title nor the abstract mentioned any DoA indicator.

Conclusion
No appropriate DoA indicator is currently available for pigs and minipigs, and welfare during anaesthesia cannot be fully ensured. Validation studies should be performed on already available and new DoA indicators, in order to provide valid and reliable tools to the scientific community, to be applied in experimental settings, especially during invasive procedures.